We thank Alejandro Brun from CISA-INIA for providing anti-Rift Valley fever Virus sera. Work in our laboratories is supported by Forschungsf&#246;rderung gem. &#167;2 Abs. 3 Kooperationsvertrag Universit&#228;tsklinikum Giessen und Marburg, the Leibniz Graduate School for Emerging viral diseases (EIDIS), the DFG Sonderforschungsbereich (SFB) 1021, and the DFG Schwerpunktprogramm (SPP) 1596.

1. Seeding of A549 Cells for Infection
<ol>
	<li>Cultivate a T75 flask of A549 cells at 37 &#176;C and 5% CO2 in cell culture medium (DMEM supplemented with 10% FCS, 526.6 mg/l L-glutamine, 50.000 U/l penicillin, and 50 mg/l streptomycin).</li>
	<li>Before starting to harvest the cells, warm up cell culture medium, PBS and 0.05% trypsin-EDTA in a waterbath heated to 37 &#176;C.</li>
	<li>Remove the medium and wash the cells with 10 ml PBS. Remove the PBS again.</li>
	<li>Add 3 ml of trypsin-EDTA and distribute equally in the flask. Transfer the flask in an incubator with 37 &#176;C and 5% CO2.</li>
	<li>When all cells are detached, add 7 ml of cell culture medium, resuspend the cells, and transfer the cell suspension into a 15 ml Falcon tube.</li>
	<li>Centrifuge the cells at 800 x g for 5 min at RT, remove the supernatant and resuspend the pellet in 10 ml fresh cell culture medium.</li>
	<li>Count the cells with a counting chamber.</li>
	<li>Add 2.5 x 106 cells in 5 ml of cell culture medium in two T25 flasks each. Incubate for 16 hr at 37 &#176;C and 5% CO2. One flask serves for the mock control and one for Cl 13 infection.</li>
</ol>
2. Infection with Rift Valley Fever Virus Clone 13 (Cl 13)
<ol>
	<li>NOTE: Cl 13 is an attenuated virus mutant which in Germany can be handled under BSL-2 conditions. Please refer to relevant national guidelines. Other typical IFN inducers would be Sendai virus (strain Cantell) or Newcastle disease virus.</li>
	<li>Pre-warm PBS, serum-free medium, and cell culture medium containing 5% FCS.</li>
	<li>Prepare 1.25 x 107 PFU/ml of Cl 13 in serum-free medium to infect 2.5 x 106 A549 cells with a multiplicity of infection (MOI) of 5. Prepare a slightly (roughly 10%) greater amount than needed to account for pipette errors.</li>
	<li>Wash the cells with PBS as described under 1.3.</li>
	<li>Add 1 ml of the Cl 13 dilution or of serum-free medium (uninfected control, mock) to the cells, and incubate for 1 hr at 37 &#176;C and 5% CO2. Move the flask carefully every 15 min to ensure equal distribution of Cl 13 dilution and serum-free medium, respectively.</li>
	<li>After 1 hr of infection, remove the inocula, add 5 ml of pre-warmed cell culture medium with 5% FCS, and incubate for 5 hr at 37 &#176;C and 5% CO2.</li>
</ol>
3. Preparation of Cell Lysates
<ol>
	<li>Prepare PBS / 0.5% Triton X-100 at 4 &#176;C. Do not add serine protease inhibitors.</li>
	<li>Wash the cells with cold PBS and add 10 ml of fresh PBS.</li>
	<li>Scrape the cells off, transfer the cell suspension in a falcon tube, and centrifuge at 800 x g for 5 min at RT.</li>
	<li>Remove the supernatant and resuspend the cell pellet in 30 &#181;l PBS / 0.5% Triton X-100. Transfer the lysate into a fresh 1.5 ml tube and incubate for at least 10 min at 4 &#176;C.</li>
	<li>Centrifuge the lysate at 10.000 x g for 10 min at 4 &#176;C and transfer the clarified cell lysate (supernatant) into a fresh tube.</li>
	<li>Determine the protein concentration by Bradford assay as described elsewhere33.</li>
	<li>Store at -20 &#176;C or proceed to trypsin digestion (4.1) or native PAGE (5.1).</li>
</ol>
4. Determination of Conformational Changes of Pattern Recognition Receptors
<ol>
	<li>TPCK-trypsin Treatment of Cell Lysates
	<ol>
		<li>Dilute L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated (TPCK) trypsin in PBS to a final working concentration of 2 &#181;g/&#181;l.</li>
		<li>Adjust in two new tubes a final protein concentration of 25 &#181;g of each protein lysate (mock or Cl 13) in a final volume of 9 &#181;l with PBS. Hence, it should be four tubes with 25 &#181;g lysate each, two times mock and two times Cl 13 infection. One set as input control (untreated) and one set for treatment with TPCK-trypsin.</li>
		<li>Add 1 &#181;l of PBS (untreated) or 1 &#181;l of 2 &#181;g/&#181;l TPCK-trypsin (final concentration: 0.2 &#181;g/&#181;l) to the cell lysates and mix the reactions by pipetting. DO NOT freeze and thaw TPCK-trypsin aliquots, because it will compromise the efficiency of digestion.</li>
		<li>Incubate the lysates at 37 &#176;C for 25 min. Stop the reaction by adding 5x denaturing sample buffer (250 mM Tris-HCl pH 6.8, 10% SDS, 50% glycerol, 25% &#946;-mercaptoethanol, 0.5% bromphenol blue) and by boiling for 5 min at 95 &#176;C. It is important to NOT extend the trypsin incubation time. In case no protease-resistant fragments are detectable, the time of trypsin digestion must be shortened.</li>
		<li>After boiling, the samples can be stored at -20 &#176;C.</li>
	</ol>
	</li>
	<li>SDS Polyacrylamide Gel Electrophoresis (PAGE) and Western Blotting
	<ol>
		<li>Load the samples on a sodium dodecyl sulfate (SDS) polyacrylamide gel containing a 5% stacking over a 12% resolving gel. Separate the proteins at 25 mA per gel until the bromphenol blue runs out.</li>
		<li>Activate a polyvinylidene fluoride (PVDF) membrane for 30 sec with methanol and put it into transfer buffer (48 mM Tris, 39 mM glycin, 1.3 mM SDS, 20% methanol).</li>
		<li>Prepare the blotting with a semidry blotting system and allow the transfer of the proteins at 10 V for 1 hr. Take the membrane out, rinse it briefly with water, and let it dry.</li>
		<li>Reactivate the membrane by shortly transferring into methanol. Wash for 5 min with TBS. Block with 10% skim milk in TBS for 1 hr at RT or at 4 &#176;C O/N. Wash the membrane 3x for 5 min each with TBS.</li>
		<li>Prepare antibody dilution as recommended in table 1 and incubate the membrane for 1 hr at RT or at 4 &#176;C O/N.</li>
		<li>Wash the membrane 3x for 10 min each with TBS-T. Add the appropriate secondary antibody coupled with horseradish peroxidase at a 1:20,000 dilution in 1% skim milk in TBS. Incubate 45 min at RT.</li>
		<li>Wash the membrane 3x for 10 min each with TBS-T, and one additional time with TBS.</li>
		<li>For signal detection use a commercial chemiluminescense kit and a digital gel imaging system.</li>
	</ol>
	</li>
	<li>Coomassie Brilliant Blue G-250 Staining
	<ol>
		<li>Perform SDS-PAGE as described in 4.2.1. Load samples on an SDS polyacrylamide gel and run the gel at 25 mA per gel until bromphenol blue runs out.</li>
		<li>Perform Coomassie Brilliant Blue G-250 staining at RT. Do all incubations under constant shaking.</li>
		<li>Transfer the gel to the fixation solution containing 40% methanol and 10% acetic acid for 30 min.</li>
		<li>Exchange the buffer to destaining solution (25% ethanol and 8% acetic acid) and incubate for 5 min.</li>
		<li>Stain the gel with 0.2% Coomassie Brillant Blue G-250 in 40% methanol and 10% acetic acid for 1 hr.</li>
		<li>Destain the gel with the destaining solution and exchange the buffer after 10 min, 30 min, and 60 min.</li>
		<li>Store the gel in 25% ethanol, 8% acetic acid, and 4% glycerol at 4 &#176;C.</li>
		<li>Perform imaging and analysis as described under 4.2.8.</li>
	</ol>
	</li>
</ol>
5. Analysis of Oligomeric States of Pattern Recognition Receptors
<ol>
	<li>Native PAGE
	<ol>
		<li>Prepare 50 &#181;g of cell lysate in a final volume of 10 &#181;l with PBS and add 5&#215; native sample buffer (250 mM Tris-HCl pH 6.8, 1% sodium deoxycholate, 50% glycerol, 0.5% bromphenol blue) to a final concentration of 1x.</li>
		<li>Load the samples IMMEDIATELY on a native polyacrylamide gel with 5% as stacking and 8% as resolving gel. Any delay will result in a loss of native complexes34.</li>
		<li>Run the gel at 20 mA per gel at 4 &#176;C with 50 mM Tris-NaOH pH 9.0, 384 mM glycin as as anode and 50 mM Tris pH 8.3, 384 mM glycin, 1% sodium deoxycholate as cathode buffer. After 1.5-2 hr (bromphenol blue band has left the gel approximately 45 min earlier) the electrophoresis is finished.</li>
	</ol>
	</li>
	<li>Western Blotting
	<ol>
		<li>Activate a polyvinylidene fluoride (PVDF) membrane for 30 sec with methanol and put it into Towbin buffer (25 mM Tris, 192 mM glycin, 0.1% SDS, 20% methanol).</li>
		<li>Assemble a wet blot chamber according to the manufacturer&#8217;s instructions and fill the tank with Towin buffer.</li>
		<li>Perform the blotting with 250 mA for 1.5 hr at 4 &#176;C.</li>
		<li>When blotting is finished proceed as described from 4.2.3 on.</li>
	</ol>
	</li>
</ol>

No conflicts of interest declared.

Sensing the presence of viruses and activation of the antiviral type I IFN system are crucial for successful innate immune responses22. Virus detection is thereby mediated by pathogen recognition receptors (PRRs) like RIG-I and PKR, enabling a rapid response and activation of antiviral defense mechanisms. Here, we describe two methods to directly evaluate the activation status of RIG-I and PKR.
Limited protease digestion as a tool to monitor conformational changes of RIG-I and PKR was first described by the groups of M. Gale Jr. and T. Fujita6,17, and J. L. Cole30, respectively. It represents a sensitive method to evaluate sensitivity alterations to trypsin treatment caused by conformational changes. Applying trypsin digestion, we detected rapid degradation of RIG-I and PKR in mock infected samples, whereas trypsin treatment of virus-infected cell lysates led to trypsin resistant RIG-I fragments. Similarly, a trypsin resistant PKR fragment was detected upon Cl 13 infection. This was accompanied by PKR phosphorylation at Thr 446, a widely used marker of PKR activation. Comparing trypsin digestion of mock and Cl 13 cell lysates by Coomassie staining demonstrates a comparable decrease of global protein levels. This indicates that formation of resistant fragments is specific for proteins like RIG-I and PKR.
The formation of oligomeric complexes of RIG-I, PKR and IRF-3 was monitored by native PAGE. Subjecting Cl 13-infected cell lysates to native PAGE, we detected RIG-I, PKR and IRF-3 oligomers, whereas these proteins remained as monomers in mock infected samples. RIG-I needs to form oligomers to activate downstream pathways. It was hypothesized that RIG-I oligomerization supports recruitment of cofactors to form a signaling platform for antiviral response mechanisms11. The function of PKR dimerization is not entirely understood. Most likely, the PKR subunits in the dimer phosphorylate each other25. The type I IFN system is tightly regulated on a transcriptional level, and IRF-3 represents one central transcription factor for the induction of IFN and ISGs38. Central hallmarks of activation are phosphorylation, dimerization, and translocation to the nucleus, where it recruits the transcriptional co-activators p300 and CREB-binding protein (CBP) to initiate IFN mRNA synthesis21,39. Analysis of IRF-3 dimerization is a widely used tool to monitor activation of the type I IFN response38. Therefore, we used the detection of IRF-3 oligomeric complexes as a proof of principle for the oligomerization assays of RIG-I and PKR oligomerization. Indeed, allowing separation of protein lysates under non-denaturing conditions, we were able to detect IRF-3 dimerization in Cl 13 infected samples.
Undoubtedly, each lab will need to optimize the protocols of limited protease digestion and native PAGE. If confronted with no detection of any resistant proteins or too many resistant fragments, one might shorten or prolong the time of digestion, respectively. Differences can also be due to the specific TPCK-trypsin stock, as preparations slightly differ. Therefore, various TPCK-trypsin concentrations should be tested for optimization. Cell lysates can be prepared from other cell lines than A549, but adaptations of the protocol might be required. It is recommended to adjust the amount of total proteins for trypsin digestion and native PAGE according to the expression level of the protein of interest in this specific cell type. Moreover, limited detection or weak separation of oligomeric complexes by native PAGE can have several reasons and can be addressed as followed: make sure that the equipment is cleaned to remove remaining denaturing agents, keep all samples and the gel during the PAGE at 4 &#176;C, and do not extend the time between sample preparation and loading onto the gel. Limited protease digestion and native PAGE have also been employed to monitor the activation of another important, closely related PRR, MDA540-42. Monitoring of MDA5 activation required different experimental conditions compared to RIG-I and PKR, and was therefore not included here.
In summary, point-by-point protocols for two useful and sensitive methods to measure activation of RIG-I and PKR are presented. Limited protease digestion and native PAGE, both coupled to Western blot analysis, permit monitoring of conformational changes and oligomerization, respectively. Using these methods, we had previously shown that RIG-I can be activated by nucleocapsids of various viruses directly after their entry into the cells32.
The exact nature and origin of the RNA species relevant for RIG-I and PKR activation in infected cells are still not entirely solved. Furthermore, many viruses interfere with the functions of PRRs by a wide variety of strategies12,43-46. The presented techniques enlarge the spectrum of methods by allowing an easy and direct measurement of RIG-I and PKR activation, are quick to perform, and do not require expensive equipment.

A crucial event in antiviral host defense is the rapid detection of the pathogen by the so-called pattern recognition receptors (PRRs)1,2. Intracellular detection of RNA virus infection is dependent on two cytoplasmic RNA helicases, RIG-I (retinoic acid inducible gene I) and MDA5 (melanoma differentiation associated protein 5)3-5. RIG-I is composed of two N-terminal caspase recruitment domains (CARDs), a central DECH-box type RNA helicase domain, and a C-terminal domain (CTD)4,6. Whereas the CTD and the helicase domain are required for recognition of non-self (viral) RNAs, the CARDs mediate downstream signaling leading to establishment of an antiviral host status.
If RIG-I is in the silent state, i.e. in the absence of a specific RNA ligand, the second CARD interacts with the central helicase domain and keeps RIG-I in an auto-inhibitory conformation7-11. RIG-I binds to short double-strand (ds) RNA bearing a 5&#8217;-triphosphate (5&#8217;PPP), long dsRNA, and polyU/UC-rich RNA, classic signature structures which are present on the genomes of many RNA viruses12-16. Two major characteristics of RIG-I activation are a switch to a closed conformation6,17 and the homo-oligomerization6,18,19. The conformational switch enhances RNA binding, exposes the CARDs for downstream signaling, and reconstitutes an active ATPase site8,9,11,20. The formation of oligomeric RIG-I complexes leads to enhanced recruitment of downstream signaling adaptor molecules to form a platform for antiviral signal transduction11. The RIG-I-regulated signaling chain eventually activates the transcription factor IRF-3 for up-regulation of interferon (IFN-alpha/beta) genes and hence the gene expression of interferon stimulated genes (ISGs) for a full antiviral response21,22. One of the best characterized ISGs is the RNA-activated protein kinase (PKR) 23. PKR belongs to the family of eukaryotic translation initiation factor 2 alpha (eIF2&#945;) kinases and is composed of an N-terminal double-stranded RNA binding domain and a C-terminal kinase domain. The kinase domain constitutes the dimerization interface crucial for PKR activation and carries out the catalytic functions of the protein. Binding of PKR to viral dsRNA leads to its conformational change permitting dimerization and auto-phosphorylation at Thr 446 among other residues. PKR then mediates phosphorylation of eIF2&#945;, thereby blocking the translation of viral mRNAs23-27.
Both RIG-I and PKR undergo major structural rearrangements, form oligomeric complexes and are post-translationally modified by phosphorylation/dephosphorylation and ubiquitination10,11,19,23,24,26-29. For a better understanding of which viral RNA structures are activating RIG-I and PKR (and at what stage viral antagonists could be interfering), it is important to precisely determine the activation status. For both PRRs it was previously described that activation leads to the emergence of trypsin-resistant protein fragments6,17,30 and higher-order oligomers6,18,19. However, given the wealth of literature on these key factors of the antiviral host response1,2,24, application of direct methods seems comparatively rare. In the hope of stimulating broader usage, we provide convenient and sensitive protocols to robustly analyze the activation states of RIG-I and PKR. The IFN competent human cell line A549 is infected with an established activator of RIG-I and PKR, the attenuated Rift Valley fever virus mutant clone 13 (Cl 13)31,32. After a simple lysing procedure, the extracts of infected cells are tested by limited trypsin digestion/western blot analysis to evaluate conformational switching, and by blue native polyacrylamide gel electrophoresis (PAGE) /Western blot analysis to measure formation of oligomers.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Cell culture&lt;/strong&gt;</td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (&lt;font class=&quot;font6&quot;&gt;DMEM)&lt;/font&gt;</td><td>Gibco</td><td>21969-035</td><td></td></tr><tr><td>OptiMEM</td><td>Gibco</td><td>31985-47</td><td></td></tr><tr><td>L-glutamine</td><td>PAA</td><td>25030-024</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>PAA</td><td>15070-063</td><td></td></tr><tr><td>Fetal calf serum (FCS)</td><td>PAA</td><td>10270</td><td></td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Chemicals&lt;/strong&gt;</td></tr><tr><td>L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated (TPCK) trypsin</td><td>Sigma Aldrich</td><td>T1426</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Antibodies&lt;/strong&gt;</td></tr><tr><td>Mouse monoclonal anti-RIG-I antibody (ALME-1)</td><td>Enzo Life Sciences</td><td>ALX-804-849-C100</td><td>WB: 1:500 in 1% skim milk in TBS</td></tr><tr><td>Mouse monoclonal anti-PKR (B10)</td><td>Santa Cruz</td><td>sc-6282</td><td>WB: 1:500 in 1% skim milk in TBS</td></tr><tr><td>Rabbit monoclonal anti-P-PKR (Thr446)</td><td>Epitomics</td><td>1120-1</td><td>WB: 1:1,000 in 5% BSA in TBS</td></tr><tr><td>Rabbit polyclonal anti-IRF-3</td><td>Santa Cruz</td><td>sc-9082</td><td>WB: 1:500 in 1% skim milk in TBS</td></tr><tr><td>Rabbit monoclonal anti-P-IRF-3 (Ser386)</td><td>IBL</td><td>og-413</td><td>WB: 1:100 in 1% skim milk in TBS</td></tr><tr><td>Rabbit anti-RVFV hyperimmune serum &quot;C2&quot; (MP-12)</td><td>Kindly provided by Alejandro Brun CISA-INIA</td><td></td><td>WB: 1:2,000 in 1% skim milk in TBS</td></tr><tr><td>Mouse monoclonal anti-beta-actin (8H10D10)</td><td>Cell Signalling</td><td>3700</td><td>WB: 1:1,000 in 1% skim milk in TBS</td></tr><tr><td>Polyclonal peroxidase-conjugated goat anti-rabbit</td><td>Thermo Fisher</td><td>0031460 1892914</td><td>WB: 1:20,000 in 1% skim milk in TBS</td></tr><tr><td>Polyclonal peroxidase-conjugated goat anti-mouse</td><td>Thermo Fisher</td><td>0031430 1892913</td><td>WB: 1:20,000 in 1% skim milk in TBS</td></tr></tbody></table>

monitoring activation of the antiviral pattern recognition receptors rig-i and pkr by limited protease digestion and native page

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, the PRRs RIG-I and PKR bind to specific viral RNA ligands. This first mediates conformational switching and oligomerization, and then enables activation of an antiviral interferon response. While methods to measure antiviral host gene expression are well established, methods to directly monitor the activation states of RIG-I and PKR are only partially and less well established.
Here, we describe two methods to monitor RIG-I and PKR stimulation upon infection with an established interferon inducer, the Rift Valley fever virus mutant clone 13 (Cl 13). Limited trypsin digestion allows to analyze alterations in protease sensitivity, indicating conformational changes of the PRRs. Trypsin digestion of lysates from mock infected cells results in a rapid degradation of RIG-I and PKR, whereas Cl 13 infection leads to the emergence of a protease-resistant RIG-I fragment. Also PKR shows a virus-induced partial resistance to trypsin digestion, which coincides with its hallmark phosphorylation at Thr 446. The formation of RIG-I and PKR oligomers was validated by native polyacrylamide gel electrophoresis (PAGE). Upon infection, there is a strong accumulation of RIG-I and PKR oligomeric complexes, whereas these proteins remained as monomers in mock infected samples.
Limited protease digestion and native PAGE, both coupled to western blot analysis, allow a sensitive and direct measurement of two diverse steps of RIG-I and PKR activation. These techniques are relatively easy and quick to perform and do not require expensive equipment.

Recognition of a viral agonist by RIG-I or PKR triggers conformational switching6,17,30 and oligomerization6,18,27. We assayed these two activation markers by limited protease digestion and native polyacrylamide gel electrophoresis (PAGE), respectively.
Human A549 cells were infected with Rift Valley fever virus clone 13 (Cl 13), which is characterized by a mutation of the IFN antagonist NSs35,36. Due to the absence of functional NSs, Clone 13 strongly induces RIG-I and PKR, leading to the establishment of a robust antiviral state in the cells12,31,32,37.
Trypsin digestion of mock infected cell lysates results in a rapid degradation of RIG-I, whereas Cl 13 infection leads to the generation of a 30 kDa resistant RIG-I fragment Figure&#160;1A. Also PKR shows partial resistance to trypsin digestion in infected samples, which coincides with its phosphorylation Figure&#160;1A. To monitor efficiency and specificity of trypsin digestion, gels were stained with Coomassie Brilliant Blue G-250. Untreated samples show equal amounts of loaded proteins Figure&#160;1B. Subjecting mock and Cl 13 cell lysates to trypsin digestion leads to a comparable decrease of global protein amounts. This demonstrates that trypsin treatment has the same efficiency for mock and Cl 13-infected samples.
The formation of oligomeric complexes was assayed by native PAGE. In uninfected cells, only monomers of RIG-I and PKR were detected Figure&#160;2. As additional control we included the transcription factor IRF-3, which is present as a monomer but known to dimerize upon activation via e.g., RIG-I38. Cl 13 infection leads to a strong accumulation of RIG-I oligomeric complexes in form of a smear and of PKR and IRF-3 dimers/oligomers as a defined protein band.
These results demonstrate that limited trypsin digestion and native PAGE are useful tools to monitor conformational changes and oligomer formation of RIG-I and PKR upon infection.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51415/51415fig1highres.jpg" width="500" /> 
Figure 1.&#160;Conformational switch of RIG-I and PKR.&#160;A549 cells were mock infected or infected with Cl 13 at an MOI of 5. After 5 hr, cells were lysed in PBS supplemented with 0.5% Triton X-100 and cleared cell lysates were either left untreated or treated with trypsin. Samples were subjected to SDS PAGE followed by Western blotting (A) or Coomassie staining (B). Blots were stained against RIG-I, PKR, phosphorylated PKR (Thr 446) and against RVFV nucleoprotein (RVFV N) and beta-actin as infection and loading control, respectively.<a href="https://www.jove.com/files/ftp_upload/51415/51415fig1highres.jpg" target="_blank"> Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="3in" src="/files/ftp_upload/51415/51415fig2highres.jpg" width="300" /> 
Figure 2.&#160;Oligomerization of RIG-I, PKR, and IRF-3. Cell lysates of mock and Cl 13-infected cell lysates were subjected to native PAGE followed by Western blot analysis. Staining was performed with antibodies against RIG-I, PKR, IRF-3, and beta-actin as loading control. PKR oligomers most likely represent dimers27.&#160;<a href="https://www.jove.com/files/ftp_upload/51415/51415fig2highres.jpg" target="_blank"> Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE at JoVE.com

Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE

Innate defenses to virus infections are triggered by pattern recognition receptors (PRRs). The two cytoplasmic PRRs RIG-I and PKR bind to viral signature RNAs, change conformation, oligomerize, and activate antiviral signaling. Methods are described which allow to conveniently monitor the conformational switching and the oligomerization of these cytoplasmic PRRs.

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, ...

monitoring-activation-antiviral-pattern-recognition-receptors-rig-i

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

12.0K Views.  Philipps-University Marburg. Innate defenses to virus infections are triggered by pattern recognition receptors (PRRs). The two cytoplasmic PRRs RIG-I and PKR bind to viral signature RNAs, change conformation, oligomerize, and activate antiviral signaling. Methods are described which allow to conveniently monitor the conformational switching and the oligomerization of these cytoplasmic PRRs.

Video: Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

Measurement of BK-polyomavirus Non-Coding Control Region Driven Transcriptional Activity Via Flow Cytometry

Polyomaviruses, like the BK-polyomavirus (BKPyV), can cause severe pathologies in immunocompromised patients. However, since highly effective antivirals are currently not available, methods measuring the impact of potential antiviral agents are required. Here, a dual fluorescence reporter that allows the analysis of the BKPyV non-coding control-region (NCCR) driven early and late promoter activity was constructed to quantify the impact of potential antiviral drugs on viral gene expression via tdTomato and eGFP expression. In addition, by cloning BKPyV-NCCR amplicons which in this protocol have been exemplarily obtained from the blood-derived DNA of immunocompromised renal transplanted patients, the impact of NCCR-rearrangements on viral gene expression can be determined. Following cloning of the patient derived amplicons, HEK293T cells were transfected with the reporter-plasmids, and treated with potential antiviral agents. Subsequently, cells were subjected to FACS-analysis for measuring mean fluorescence intensities 72 h post transfection. To also test the analysis of drugs that have a potential cell cycle inhibiting effect, only transfected and thus fluorescent cells are used. Since this assay is performed in large T Antigen expressing cells, the impact of early and late expression can be analyzed in a mutually independent manner.

In this manuscript, a protocol is presented to perform FACS-based measurement of BK-polyomavirus transcriptional activity by using HEK293T cells transfected with a bidirectional reporter plasmid expressing tdTomato and eGFP. This method further allows to quantitatively determine the influence of novel compounds on viral transcription.

Polyomaviruses, like the BK-polyomavirus (BKPyV), can cause severe pathologies in immunocompromised patients. However, since highly effective antivirals ...

Genetics

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Biochemistry

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.

We describe a cell-based assay to report on HIV-1 fusion via the expression of green fluorescent protein detectable by flow cytometry or fluorescence microscopy. It can be used to test inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems.

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

Native Polyacrylamide Gel Electrophoresis Immunoblot Analysis of Endogenous IRF5 Dimerization

Interferon regulatory factor 5 (IRF5) is a key transcription factor for regulating the immune response. It is activated downstream of the Toll-like receptor myeloid differentiation primary response gene 88 (TLR-MyD88) signaling pathway. IRF5 activation involves phosphorylation, dimerization, and subsequent translocation from the cytoplasm into the nucleus, which in turn induces the gene expression of various pro-inflammatory cytokines. A detection assay for IRF5 activation is essential to studying IRF5 functions and its relevant pathways. This article describes a robust assay to detect endogenous IRF5 activation in the CAL-1 human plasmacytoid dendritic cell (pDC) line. The protocol consists of a modified nondenaturing electrophoresis assay that can distinguish IRF5 in its monomer and dimer forms, thus providing an affordable and sensitive approach to analyze IRF5 activation.

A native Western blot method for analyzing endogenous interferon regulatory factor 5 dimerization in the CAL-1 plasmacytoid dendritic cell line is described. This protocol can be applied to other cell lines as well.

Interferon regulatory factor 5 (IRF5) is a key transcription factor for regulating the immune response. It is activated downstream of the Toll-like ...

Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection

Caspases, a family of cysteine proteases, orchestrate programmed cell death in response to various stimuli, including microbial infections. Initially described to occur by apoptosis, programmed cell death is now known to encompass three interconnected pathways: pyroptosis, apoptosis, and necroptosis, together coined as one process, PANoptosis. Influence A virus (IAV) infection induces PANoptosis in mammalian cells by inducing the activation of different caspases, which, in turn, cleave various host as well as viral proteins, leading to processes like the activation of the host innate antiviral response or the degradation of antagonistic host proteins. In this regard, caspase 3-mediated cleavage of host cortactin, histone deacetylase 4 (HDAC4), and histone deacetylase 6 (HDAC6) has been discovered in both animal and human epithelial cells in response to the IAV infection. To demonstrate this, inhibitors, RNA interference, and site-directed mutagenesis were employed, and, subsequently, the cleavage or resistance to cleavage and the recovery of cortactin, HDAC4, and HDAC6 polypeptides were measured by western blotting. These methods, in conjunction with RT-qPCR, form a simple yet effective strategy to identify the host as well as viral proteins undergoing caspase-mediated cleavage during an infection of IAV or other human and animal viruses. The present protocol elaborates the representative results of this strategy, and the ways to make it more effective are also discussed.

Influenza A virus (IAV) infection activates the caspases that cleave host and viral proteins, which, in turn, have pro- and antiviral functions. By employing inhibitors, RNA interference, site-directed mutagenesis, and western blotting and RT-qPCR techniques, caspases in infected mammalian cells that cleave host cortactin and histone deacetylases were identified.

Caspases, a family of cysteine proteases, orchestrate programmed cell death in response to various stimuli, including microbial infections. Initially ...

Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-Dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells

The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical regulators of necroptosis, an inflammatory form of cell death with important antiviral functions. Autophosphorylation of RIPK3 induces phosphorylation and activation of the pore-forming executioner protein of necroptosis MLKL. Trafficking and oligomerization of phosphorylated MLKL at the cell membrane results in cell lysis, characteristic of necroptotic cell death. The nucleic acid sensor ZBP1 is activated by binding to left-handed Z-form double-stranded RNA (Z-RNA) after infection with RNA and DNA viruses. ZBP1 activation restricts virus infection by inducing regulated cell death, including necroptosis, of infected host cells. Immunofluorescence microscopy permits the visualization of different signaling steps downstream of ZBP1-mediated necroptosis on a per-cell basis. However, the sensitivity of standard fluorescence microscopy, using current commercially available phospho-specific antibodies against human RIPK3 and MLKL, precludes reproducible imaging of these markers. Here, we describe an optimized staining procedure for serine (S) phosphorylated RIPK3 (S227) and MLKL (S358) in human HT-29 cells infected with herpes simplex virus 1 (HSV-1). The inclusion of a tyramide signal amplification (TSA) step in the immunofluorescent staining protocol allows the specific detection of S227 phosphorylated RIPK3. Moreover, TSA greatly increases the sensitivity of the detection of S358 phosphorylated MLKL. Together, this method enables the visualization of these two critical signaling events during the induction of ZBP1-induced necroptosis.

Tyramide signal amplification during immunofluorescent staining enables the sensitive detection of phosphorylated RIPK3 and MLKL during ZBP1-induced necroptosis after HSV-1 infection.

The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical ...

Evaluation of Caspase Activation to Assess Innate Immune Cell Death

Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is the initiation of innate immune programmed cell death (PCD) to eliminate infected or damaged cells and propagate immune responses. However, excess PCD is associated with inflammation and pathology. Therefore, understanding the activation and regulation of PCD is a central aspect of characterizing innate immune responses and identifying new therapeutic targets across the disease spectrum.
This protocol provides methods for characterizing innate immune PCD activation by monitoring caspases, a family of cysteine-dependent proteases that are often associated with diverse PCD pathways, including&#160;apoptosis,&#160;pyroptosis, necroptosis, and PANoptosis. Initial reports characterized caspase-2, caspase-8, caspase-9, and caspase-10 as initiator caspases and caspase-3, caspase-6, and caspase-7 as effector caspases in apoptosis, while later studies found the inflammatory caspases, caspase-1, caspase-4, caspase-5, and caspase-11, drive pyroptosis. It is now known that there is extensive crosstalk between the caspases and other innate immune and&#160;cell death molecules across the previously defined PCD pathways, identifying a key knowledge gap in the mechanistic understanding of innate immunity and PCD and&#160;leading to the characterization of PANoptosis. PANoptosis is a unique innate immune inflammatory PCD pathway regulated by PANoptosome complexes, which integrate components, including caspases,&#160;from other cell death pathways.
Here, methods for assessing the activation of caspases in response to various stimuli are provided. These methods allow for the characterization of PCD pathways both in vitro and in vivo, as activated caspases undergo proteolytic cleavage that can be visualized by western blotting using optimal antibodies and blotting conditions. A protocol and western blotting workflow have been established that allow for the assessment of the activation of multiple caspases from the same cellular population, providing a comprehensive characterization of the PCD processes. This method can be applied across research areas in development, homeostasis, infection, inflammation, and cancer to evaluate PCD pathways throughout cellular processes in health and disease.

This protocol describes a comprehensive method for assessing caspase activation (caspase-1, caspase-3, caspase-7, caspase-8, caspase-9, and caspase-11) in response to both in vitro and in vivo (in mice) models of infection, sterile insults, and cancer to determine the initiation of cell death pathways, such as pyroptosis, apoptosis, necroptosis, and PANoptosis.

Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is ...